International Journal of Molecular Sciences

Review Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

Martin Bonin , Sruthi Sreekumar, Stefan Cord-Landwehr and Bruno M. Moerschbacher * Institute for Biology and Biotechnology of Plants, University of Münster, 48143 Münster, Germany; [email protected] (M.B.); [email protected] (S.S.); [email protected] (S.C.-L.) * Correspondence: [email protected]



Received: 30 September 2020; Accepted: 19 October 2020; Published: 22 October 2020


Abstract: During the past decade, detailed studies using well-defined ‘second generation’ chitosans have amply proved that both their material properties and their biological activities are dependent on their molecular structure, in particular on their degree of polymerisation (DP) and their fraction of acetylation (FA). Recent evidence suggests that the pattern of acetylation (PA), i.e., the sequence of acetylated and non-acetylated residues along the linear polymer, is equally important, but chitosan polymers with defined, non-random PA are not yet available. One way in which the PA will influence the bioactivities of chitosan polymers is their enzymatic degradation by sequence-dependent chitosan hydrolases present in the target tissues. The PA of the polymer substrates in conjunction with the subsite preferences of the hydrolases determine the type of oligomeric products and the kinetics of their production and further degradation. Thus, the bioactivities of chitosan polymers will at least in part be carried by the chitosan oligomers produced from them, possibly through their interaction with pattern recognition receptors in target cells. In contrast to polymers, partially acetylated chitosan oligosaccharides (paCOS) can be fully characterised concerning their DP, FA, and PA, and chitin deacetylases (CDAs) with different and known regio-selectivities are currently emerging as efficient tools to produce fully defined paCOS in quantities sufficient to probe their bioactivities. In this review, we describe the current state of the art on how CDAs can be used in forward and reverse mode to produce all of the possible paCOS dimers, trimers, and tetramers, most of the pentamers and many of the hexamers. In addition, we describe the biotechnological production of the required fully acetylated and fully deacetylated oligomer substrates, as well as the purification and characterisation of the paCOS products.

Keywords: paCOS; chito-oligosaccharide; CDA; chitin deacetylase; pattern of acetylation; chitosan; chitin; chitinase; chitosanase; regio-selective

1. Why Using Defined Chitosans Is So All-Important in Bioactivity Studies The term ‘chitosan’ describes a large and versatile family of oligo- and polysaccharides with different structures and multiple functions. Some chitosans have antimicrobial activity, inhibiting bacterial growth as well as germination, growth and development of fungi and oomycetes. Some chitosans have antiviral activity and some inhibit growth and development of nematodes. Some chitosans stimulate plant growth and development, some induce plant disease resistance, and some improve plant abiotic stress tolerance. Some chitosans support scar-free wound healing in animals and humans, some appear to inhibit tumour cell growth, some may have anti-inflammatory or anti-oxidant potential, and a plethora of other biomedically relevant bioactivities have also been reported. However, all of these biological functionalities have been notoriously unreliable. Sometimes, the above-mentioned bioactivities can be observed, and sometimes not. We have argued that we need to know molecular

Int. J. Mol. Sci. 2020, 21, 7835; doi:10.3390/ijms21217835 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7835 2 of 22 structure-function relationships of chitosans to achieve reliable results [1,2]. Chitosans differ in their degree of polymerisation (DP) and in their fraction of acetylation (FA), and both parameters deeply influence the physico-chemical properties and the biological activities of the polymers. Two landmark papers by Kauss et al. [3] and Vander et al. [4] represent crucial steps in this process of understanding structure-function relationships of partially acetylated chitosan polymers. Both papers clearly indicated the crucial role of FA and, less marked, of DP for the biological activity of different chitosans, as exemplified in their elicitor activities towards plant cells. Kauss et al. [3] reported that the elicitor activity of chitosan polymers with sufficiently high DP decreased with increasing FA, i.e., high-DP low-FA chitosans were most active in inducing resistance reactions in Catharanthus roseus cells. The authors concluded that it is the polycationic character of the chitosan which is responsible for the elicitor activity, and they suggested electrostatic interactions between the polycationic polymer and its suggested target, the polyanionic phospholipid membrane, as a mode of action. We might call this the ‘target hypothesis’ of chitosans’ mode of action. Vander et al. [4] reported that the elicitor activity of chitosan polymers with sufficiently high DP first increased, then decreased with increasing FA; very low-FA chitosans and very high-FA chitosans were elicitor-inactive, only high-DP medium-FA chitosans were active in inducing resistance reactions in wheat leaves. The authors concluded that it is the combination of the partly polycationic and partly hydrophobic character of the chitosan which is responsible for the elicitor activity, and they suggested molecular recognition by a most-likely transmembrane proteinaceous, partly polyanionic and partly hydrophobic receptor as a mode of action. We might call this the ‘receptor hypothesis’ of chitosans’ mode of action. Based on these and similar later results, the chitosan matrix was introduced to visualise the structure-function dependencies of partially acetylated chitosans, clearly indicating that different biological activities depend on different structural aspects of chitosans [1,5,6]. This marked the transition from yesterday’s first-generation raw chitosan to today’s second-generation defined chitosans. First-generation chitosan samples are poorly defined mixtures of molecules with highly different and mostly unknown DP and FA, hence with potentially huge batch-to-batch differences. Second-generation chitosans, in contrast, are well-defined in terms of their DP and FA and, therefore, possess defined physico-chemical properties and known biological activities. Upon dissemination of this scientific knowledge, some forward-thinking commercial chitosan producers began to introduce rigorous quality control measures so that second-generation chitosans became available in good quality and at industrial scale, allowing the development of reliable chitosan-based products. While we now know much about molecular structure-function relationships of chitosans, we are still far from understanding their cellular modes of action. The discussion of whether chitosans act by rather non-specifically interacting with target structures (i.e., the ‘target hypothesis’) or rather by specifically interacting with cognate receptor molecules (i.e., the ‘receptor hypothesis’) is ongoing. A crucial consideration here is the fact that practically all organisms appear to secrete sequence-specific chitosan hydrolases [7,8]. Most bacteria and fungi secret chitinases and/or chitosanases, and these may protect them from the antimicrobial action of chitosan polymers [9–11], arguing in favour of the membrane target hypothesis which requires polymers for bioactivity. In contrast, human, animal, and plant cells rather secret chitinases, but not chitosanases, and these may be essential in generating partially acetylated chitosan oligosaccharides (paCOS) which may be recognised by specific receptors. However, to date, no chitosan-specific receptor has been described in any detail, while chitin receptors are known in both humans and plants [12–15]. Perhaps, the observed elicitor activity of medium- FA chitosans in plants can best be explained by the action of plant chitinases degrading high-DP medium-FA chitosan polymers into low-DP low-FA chitosan polymers and high-FA chitosan oligomers, the former non-specifically reacting with target membranes and the latter being specifically recognised by chitin receptors [2]. In that case, both the target and the receptor hypotheses would apply. Thus, a picture is emerging in which sequence-specific chitosan hydrolases process partially acetylated chitosan polymers, resulting in smaller, modified chitosan polymers and yielding structurally Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 22 Int. J. Mol. Sci. 2020, 21, 7835 3 of 22

structurally more or less defined mixtures of paCOS. In this scenario, the pattern of acetylation (PA), morei.e., the or distribution less defined of acetylated mixtures ofGlcNAc paCOS. (A) Inand this de-acetylated scenario, theGlcN pattern (D) units of in acetylation the chitosan (PA), chains, i.e., theemerges distribution as a third of acetylatedand crucial GlcNAc determinant (A) and of de-acetylatedbioactivities. First, GlcN the (D) differen units int sub-site the chitosan specificities chains, emergesand preferences as a third of andthe crucialsequence-specific determinant chitosan of bioactivities. hydrolases First, for the GlcNAc different or GlcN sub-site units specificities determine and at preferenceswhich sites-if of at the all-a sequence-specific given chitosan chitosan polymer hydrolases will be cleaved, for GlcNAc and they or GlcN will concomitantly units determine determine at which sites-ifthe occurrence at all-a givenand frequency chitosan polymerof GlcNAc will and be cleaved,GlcN units and at they and willnear concomitantly the new reducing determine and non- the occurrencereducing ends and of frequency the paCOS of GlcNAc generated—if and GlcN any units [16,17]. at and And near second, the new the reducing charges andand non-reducinghydrophobic endspatches of present the paCOS at the generated—if ligand binding any site [16 of,17 the]. Andreceptors second, will thedetermine charges which and hydrophobic paCOS with patchesdefined presentPA will at bind the ligandmost efficiently binding site and, of thethus, receptors trigger will a reaction. determine Importantly, which paCOS thus, with PA defined will crucially PA will binddetermine most biological efficiently activities and, thus, of triggerchitosan a polyme reaction.rs independently Importantly, thus, of whether PA will the crucially target hypothesis determine biologicalor rather the activities receptor of chitosanhypothesis polymers (or both) independently will eventually of whetherbe verified. the target hypothesis or rather the receptorClearly hypothesis then, the (or above both) mentioned will eventually 2D matrix be verified. will need to be expanded to a 3D matrix, including the influenceClearly then, of PA the on above physico-chemical mentioned 2D properties matrix will and need biological to be expanded activities to [1,5,6]. a 3D matrix, This 3D including matrix thewill influence mark the of transition PA on physico-chemical from today’s propertiessecond-generation and biological defined activities chitosans [1,5, 6to]. Thistomorrow’s 3D matrix third- will markgeneration the transition designer from chitosans. today’s Third-generation second-generation chitosans defined will chitosans be defined to tomorrow’s not only third-generationin their DP and designerFA, but also chitosans. in their Third-generation PA. If we want chitosans to more will befully defined understand not only inmolecular their DP andstructure-functionFA, but also in theirrelationships PA. If we and want cellular to more modes fully understandof action for molecular each of structure-functionthe many biological relationships activities andreported cellular or modessuggested of action in literature, for each we of thewill many need biologicalfully defined activities paCOS, reported i.e., paCOS or suggested with known in literature, DP, FA, and wewill PA need(Figure fully 1) defined[18]. Such paCOS, paCOS i.e., can paCOS best with be produced known DP, usingFA, and regio-selective PA (Figure1 )[chitin18]. Suchdeacetylases, paCOS can as bestdescribed be produced in this review. using regio-selective chitin deacetylases, as described in this review.

Figure 1.1. Partially acetylated chito-oligosaccharides (p (paCOS)aCOS) are characterised by three structuralstructural parameters, namelynamely (i)(i) the degree of polymerisation (DP),(DP), i.e., the number of monomeric units, (ii) the fraction of acetylation (FA), i.e., the relative abundance of GlcNAc units, and (iii) the pattern of fraction of acetylation (FA), i.e., the relative abundance of GlcNAc units, and (iii) the pattern of acetylation (PA), i.e., the sequence of GlcNAc and GlcN units. acetylation (PA), i.e., the sequence of GlcNAc and GlcN units. 2. Regio-Selective Chitin de-N-Acteylases 2. Regio-Selective Chitin de-N-Acteylases Chitin deacetylases (CDAs) catalyse the hydrolytic cleavage of the acetamido bond in GlcNAc unitsChitin within deacetylases chitin and chitosan(CDAs) catalyse oligomers the and hydrolyt polymers,ic cleavage deacetylating of the acetamido them to yieldbond GlcNin GlcNAc units. Theyunits belongwithin tochitin carbohydrate and chitosan esterase oligomers family CE4,and andpolymers, their three-dimensional deacetylating them structures to yield as GlcN well asunits. the modeThey belong of their to catalytic carbohydrate reaction estera havese been family described CE4, and in several their three-di recent reviewsmensional [19– 22structures]. Like many as well other as polysaccharidethe mode of their modifying catalytic enzymes,reaction have such been as chitinases described and in several chitosanases recent [ 16reviews,17,23– [19–22].25], CDAs Like possess many aother substrate polysaccharide binding site modifying consisting enzymes, of several such subsites, as chitinases each of whichand chitosanases binds a single [16,17,23–25], monosaccharide CDAs unitpossess of the a substratesubstrate [26binding–28]. The site catalytic consisting subsite, of several i.e., the subsites, one binding each the of GlcNAc which unitbinds that a willsingle be deacetylatedmonosaccharide to yield unit aof GlcN the substrate unit, is defined [26–28]. as The subsite catalytic {0}, thesubsite, subsite(s) i.e., the ‘left’ one of binding it, i.e., towardsthe GlcNAc the non-reducingunit that will endbe deacetylated of the substrate, to yield are numbered a GlcN unit, as { is1}, defined { 2}, etc., as subsite while those {0}, the ‘right’ subsite(s) of subsite ‘left’ {0}, of i.e., it, − − towardsi.e., towards the reducingthe non-reducing end of the end substrates, of the substrate, are {+1} are etc. numbered (Figure2)[ as 26{−].1}, Crystal {−2}, etc., structures while those of several ‘right’ fungalof subsite CDAs {0}, suggesti.e., towards that their the reducing substrate end binding of th sitee substrates, comprises are four {+1} subsites, etc. (Figure ranging 2) [26]. from Crystal { 2} to − {structures+1} [20,28 ,of29 ],several while somefungal bacterial CDAs suggest CDAs appearthat their to havesubstrate less subsitesbinding [site29], comprises and one insect four CDAsubsites, has beenranging shown from to {− possess2} to {+1} a larger[20,28,29], number while of some subsites bacterial [30]. CDAs appear to have less subsites [29], and one insect CDA has been shown to possess a larger number of subsites [30].

Int. J. Mol. Sci. 2020, 21, 7835 4 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 22

LikeLike in in any any other other enzyme, enzyme, thethe molecularmolecular finefine structurestructure of the substrate substrate binding binding site site of of a aCDA CDA determinesdetermines its its substrate substrate specificity specificity oror preference.preference. Tw Twoo didifferentfferent structural feat featuresures appear appear to to govern govern thethe mode mode of of substrate substrate binding binding inin CDAs,CDAs, andand thusthus theirtheir regio-selectivity, namely namely the the position position and and flexibilityflexibility of of loops loops surrounding surroundingthe the bindingbinding site,site, andand the specificity specificity or or preference preference for for GlcNAc GlcNAc versus versus GlcNGlcN binding binding at at the the di differentfferent subsites. subsites. TheThe subsitesubsite capping model, which which was was developed developed based based on on a a seriesseries of of crystal crystal structures structures of of the theVibrio Vibrio cholerae choleraeCDA CDA in in di differentfferent states states of of substratesubstrate binding,binding, describesdescribes a seta ofset six of loopssix loops surrounding surrounding the the active active site site and and suggests suggests an inducedan induced fit modelfit model for for substrate substrate binding binding [29 ]. These[29]. loopsThese determineloops determine the preferred the preferred positioning positionin of theg of substrate the substrate within within the binding the binding site. This site. bringsThis thebrings different the different subunits subunits of the substrate of the substrate into close into proximity close proximity to the amino to the acids amino forming acids theforming substrate the bindingsubstrate cleft, binding including cleft, including the catalytically the catalytically active amino active acid amino residues acid residues at subsite at subsite {0}. A partial{0}. A partial or full or full negative charge in the substrate binding cleft may favour, a positive one disfavour the binding negative charge in the substrate binding cleft may favour, a positive one disfavour the binding of a of a GlcN unit at that particular position, while the binding of a GlcNAc unit can be expected to GlcN unit at that particular position, while the binding of a GlcNAc unit can be expected to require require more space and to be supported by the presence of a hydrophobic pocket to accommodate more space and to be supported by the presence of a hydrophobic pocket to accommodate the terminal the terminal methyl group of the acetamido side chain. In fact, the first chitosan (rather than chitin) methyl group of the acetamido side chain. In fact, the first chitosan (rather than chitin) deacetylase has deacetylase has recently been described, which favours GlcN over GlcNAc at subsite {−1}, and thus recently been described, which favours GlcN over GlcNAc at subsite { 1}, and thus partially acetylated partially acetylated chitosan over chitin as a substrate [27]. − chitosan over chitin as a substrate [27].

FigureFigure 2. 2.Fungal Fungal chitin chitin deacetylases deacetylases (CDA) (CDA) (right), (right), which which are are better better known known than than e.g., e.g., bacterial bacterial (left) (left) or insector insect CDAs, CDAs, typically typically possess possess four subsitefour subsite binding binding sites (ranging sites (ranging from {–2} from to {{–2}+1}) to within {+1}) awithin substrate a bindingsubstrate cleft, binding highlighted cleft, highlighte in beige,d each in beige, of which each bindsof which one binds monosaccharide one monosaccharide subunit of subunit the substrate, of the a chitinsubstrate, or chitosan a chitin oligomeror chitosan or polymer.oligomer Eachor polymer. subsite Each can havesubsite its owncan have specificity its own or preferencespecificity or for bindingpreference a N -acetylglucosaminefor binding a N-acetylglucosamine or a glucosamine or unit.a glucosamine The substrate unit. binding The substrate cleft, shown binding in cleft, beige, mayshown be delineated in beige, may by sixbe proteindelineated loops by (L1six protein to L6) which loops may(L1 to be L6) flexible, which allowingmay be flexible, an induced allowing fit of an the enzyme.induced Left, fit of schematic the enzyme. representation Left, schematic of therepresentation bacterial VcCDA of the bacterial from Vibrio VcCDAfor which from Vibrio the surrounding for which loopsthe wheresurrounding first described; loops where right, firs 3D-modelt described; of the right, fungal 3D-model ClCDA fromof the the fungalColletotrichum ClCDAwith from bound the substrate,Colletotrichum chitin with tetraose. bound substrate, chitin tetraose.

RecentRecent advances advances inin thethe heterologousheterologous expressionexpression of genes coding coding for for CDAs CDAs [31] [31 ]and and in in the the quantitativequantitative mass mass spectrometric spectrometric sequencingsequencing of paCOS [32] [32] are currently leading leading to to an an increasing increasing numbernumber of of CDAs CDAs with with known known subsite subsite bindingbinding preferencespreferences and, hence, known known products products when when acting acting onon fully fully acetylated acetylated chitinchitin oligomers (An) (An) as as substr substratesates (Table (Table 1).1 ).These These enzymes enzymes can canbe used be used for the for theproduction production of ofthe the fully fully defined defined paCOS paCOS required required for for advancing advancing our our understanding understanding of of molecular molecular structure-functionstructure-function relationships relationships and and cellular cellular modes modes of action of action of biologically of biologically active, partially active, acetylatedpartially chitosans.acetylated The chitosans. portfolio The of portfolio defined paCOSof defined that paCOS can be that produced can be usingproduced CDAs using has CDAs been drasticallyhas been increaseddrastically with increased the realisation with the that realisation these enzymes that these can enzymes also be usedcan also to catalyse be used theto catalyse reverse reaction,the reverse i.e., thereaction,N-acetylation i.e., the of N fully-acetylation de-acetylated of fully glucosamine de-acetylated oligomers glucosamine (Dn) oligomers in the presence (Dn) in of the excess presence amounts of ofexcess acetate, amounts while mainlyof acetate, retaining while mainly their regio-selectivity retaining their regio-selectivity [33,34]. [33,34]. MostMost of of the the times, times, however, however, thethe paCOSpaCOS resultingresulting from CDA action on on chitin chitin or or glucosamine glucosamine oligomersoligomers will will not not be be entirely entirely pure pure so that so that a final a chromatographicfinal chromatographic purification purification step is step typically is typically needed.

Int. J. Mol. Sci. 2020, 21, 7835 5 of 22

In the following paragraphs, we will first describe the production of the substrates, i.e., fully acetylated chitin and fully deacetylated glucosamine oligomers of defined DP,followed by the enzymatic strategies that can yield fully defined dimeric to hexameric paCOS. The chapter will end with short descriptions of the state-of-the-art of paCOS analysis and purification.

Table 1. Products of regio-selective CDAs which have been characterized in detail.

Substrate Enzyme Step Reference AA AAA AAAA AAAAA AAAAAA NodB 1 DA DAA DAAA DAAAA DAAAAA [35] VcCDA 1 AD ADA ADAA ADAAA ADAAAA [36] CnCDA4 1 - (ADA) AADA AAADA AAAADA [27] 2 - AADD AADDA AAADDA 3 - - AADDDA PesCDA 1 - - AADA AA[AD]A AA[A2D1]A [37] 2 - AADDA AA[A1D2]A 3 - AADDDA AAADA AAADAA PgtCDA 1 p.n.d. p.n.d. AADA [31] (AADAA) (AADAAA) AADDA AAADDA 2 AADD (AAADD) (AADDAA) AADDDA 3 - AADDD (AAADDD) 4 - AADDDD PcCDA 1 a.n.d. - A3D1 ADAAA a.n.d. [38] 2 A2D2 ADDAA ADAA AAADA BsPdaC-CD 1 - p.n.d. a.n.d. [39] AADA (AADAA) ADDA AADDA 2 (DADA) (ADADA) 3 DDDA ADDDA 4 - DDDDA () = by-product; p.n.d. = pattern not determined; a.n.d. = activity not determined; [] = pattern not completely resolved; - = no activity; A = GlcNAc; D = GlcN.

3. Preparation of the Substrates (An and Dn) The enzymatic production of defined paCOS using CDAs depends on the availability of suitably pure substrates, namely fully acetylated chitin oligomers and fully deacetylated glucosamine oligomers of defined DP. Different methods to this end have been described, and such oligomers are commercially available, though at rather high prices only, due to the rather expensive production and purification steps involved. One approach is the partial acid or enzymatic hydrolysis of chitin to yield chitin oligomers (An), or of polyglucosamine to yield glucosamine oligomers (Dn) (Figure3). Chitin is commercially available, and it can be converted to polyglucosamine using sequential steps of alkaline treatment [40,41]. Clearly, the purity of the polymeric substrates will determine the product quality that can be achieved. Acid hydrolysis of chitin needs to be performed carefully, e.g., using concentrated rather than dilute acids, to prevent concomitant loss of acetyl groups [42–44]. Enzymatic depolymerisation of chitin and polyglucosamine can be achieved using chitinases and chitosanases, respectively. Care must be taken to avoid the use of processive enzymes which would yield mostly dimeric products [23,45]. The main drawback of this approach is that both chemical and enzymatic hydrolyses are difficult to control, both proceed quickly to the production of very small, mostly monomeric (acid hydrolysis) or di- and trimeric (enzymatic hydrolysis) products [46]. Int. J. Mol. Sci. 2020, 21, 7835 6 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 22

FigureFigure 3.3. Chemo-enzymaticChemo-enzymatic productionproduction ofof fullyfully acetylatedacetylated chitinchitin oligomersoligomers andand fullyfully de-acetylatedde-acetylated glucosamineglucosamine oligomers.oligomers. Fully Fully acet acetylatedylated chitin dimers A2 and trimers A3 can bebe generatedgenerated fromfrom polymericpolymeric chitinchitin byby thethe actionaction ofof aa chitinase,chitinase, fullyfully de-acetylatedde-acetylated dimersdimers D2D2 andand trimerstrimers D3D3 fromfrom polyglucosamine,polyglucosamine, itself preparedprepared fromfrom chitinchitin byby sequentialsequential stepssteps ofof alkalinealkaline de-acetylation,de-acetylation, byby thethe actionaction ofof aa chitosanase.chitosanase. Larger oligomersoligomers cancan bebe producedproduced byby thethe actionaction ofof chitinasechitinase oror chitosanasechitosanase onon partiallypartially acetylatedacetylated chitosanchitosan polymerspolymers followedfollowed byby alkalinealkaline de-acetylationde-acetylation yieldingyielding GlcNGlcN oligomers,oligomers, oror byby chemicalchemical NN-acetylation-acetylation usingusing aceticacetic anhydride,anhydride, yieldingyielding GlcNAcGlcNAc oligomers.oligomers.

AnAn alternativealternative approachapproach givinggiving moremore easyeasy accessaccess toto largerlarger oligomersoligomers isis thethe partialpartial acidacid oror enzymaticenzymatic hydrolysishydrolysis of partiallyof partia acetylatedlly acetylated chitosan chitosan polymers polymers followed followed by fullchemical by full Nchemical-acetylation N- toacetylation yield chitin to oligomersyield chitin (An) oligomers or by full (An) chemical or by de-acetylationfull chemical tode-acetylation yield glucosamine to yield oligomers glucosamine (Dn). Theoligomers choice (Dn). of polymeric The choice substrate of polymeric used, substrate particularly used, its particularlyFA, and of theits F depolymerisingA, and of the depolymerising agent used, particularlyagent used, itsparticularly sequence specificity,its sequence will specificity, determine wi thell determine size range ofthe products size range obtained. of products Theglycosidic obtained. linkageThe glycosidic of a GlcNAc linkage residue of a GlcNAc within a residue chitosan within polymer a chitosan is more acid-labilepolymer is thanmore that acid-labile of a GlcN than unit that so thatof a theGlcN polymer unit so chain that the will polymer preferentially chain will break preferentially at a GlcNAc break residue at duringa GlcNAc acid residue hydrolysis during [42 acid,44]. Consequently,hydrolysis [42,44]. the size Consequently, of the oligomeric the size products of the oligomeric obtained, which productscum granoobtained, salis whichrepresent cum contiguous grano salis D-blocksrepresent withincontiguous the original D-blocks polymer within the chain, original will increasepolymer withchain, decreasing will increaseFA withof the decreasing chitosan used.FA of Inthe fact, chitosan as the used. distribution In fact, ofas GlcNActhe distribution units within of GlcNAc commercial units chitosanswithin commercial follows a randomchitosans (Bernoulli) follows a distributionrandom (Bernoulli) [47,48], distribution the size range [47,48], of oligomeric the size range products of oligomeric obtained products can roughly obtained be predicted can roughly as abe function predicted of as the a FfunctionA of the of polymer the FA used.of the However,polymer used. as chitosan However, polymer as chitosan samples polymer invariably samples are mixturesinvariably of are molecules mixtures with of molecules different F Awithso thatdifferent the FA FAgiven so that or the measured FA given is anor averagemeasured value is an only average [49], withvalue unknown only [49], dispersity with unknownÐFA [ 6dispersity], such a calculationĐFA [6], such can a calculation give a rough can estimate give a rough only of estimate the true only DP rangeof the experimentallytrue DP range experimentally obtained. obtained. TheThe hydrolytichydrolytic processprocess cancan bebe controlledcontrolled moremore preciselyprecisely when using enzymes instead of acid. ChitinasesChitinases requirerequire atat leastleast oneone GlcNAcGlcNAc unitunit withinwithin thethe chitosanchitosan polymerpolymer chainchain toto bindbind andand hydrolysehydrolyse theirtheir substratesubstrate [[50–53],50–53], whilewhile chitosanaseschitosanases requirerequire atat leastleast oneone GlcNGlcN unitunit [[17,25].17,25]. Hence,Hence, thethe sizesize ofof chitinasechitinase productsproducts increases,increases, whilewhile thatthat ofof chitosanasechitosanase productsproducts decreasesdecreases withwith decreasingdecreasingF FAA ofof thethe substrate.substrate. While the DP range of th thee products is more difficult difficult to pr predictedict compared to acid hydrolysis, unlessunless thethe subsitesubsite specificityspecificity ofof thethe enzymeenzyme usedused isis preciselyprecisely known,known, enzymaticenzymatic hydrolysishydrolysis cancan moremore easilyeasily yield a broader broader range range of of prod products,ucts, as as enzymes enzymes with with different different levels levels of subsite of subsite specificities specificities are areavailable available [17,32,54–56]. [17,32,54– 56Clearly,]. Clearly, the more the morespecific specific the enzyme, the enzyme, the larger the its larger products. its products. In any case, In any the case,enzymatic the enzymatic products products will be willmixtures be mixtures of partia of partiallylly acetylated acetylated oligomers oligomers (paCOS) (paCOS) which which can can be beconverted converted to tofully fully acetylated acetylated chitin chitin oligomers oligomers (An) (An) by by NN-acetylation-acetylation or or to fullyfully de-acetylatedde-acetylated glucosamineglucosamine oligomersoligomers (Dn)(Dn) byby de-acetylation,de-acetylation, asas above.above. Certainly,Certainly, thethe mostmost elegant,elegant, butbut alsoalso thethe mostmost demandingdemanding approachapproach towardstowards productionproduction ofof chitinchitin oligomersoligomers (An)(An) is is their their biotechnological biotechnological production production in in dedicated dedicated cell cell factories factories (Figure (Figure4)[ 4)57 [57].]. As As early early as 1994,as 1994, Geremia Geremia et al. et expressed al. expressed a bacterial a bacterial chitin oligomer chitin oligomer synthase, synthase, NodC from NodCAzorhizobium from Azorhizobium caulinodans, incaulinodansE. coli, driving, in E. the coli, synthesis driving ofthe chitin synthesis pentamer of chitin (A5) pentamer [58]. The beauty(A5) [58]. of this The approach beauty of is this its scalability: approach gramis its scalability: amounts of gram rather amounts pure chitin of rather oligomers pure chitin can thusoligomers easily can be thus produced easily [be59]. produced Co-expressing [59]. Co- a expressing a chitinase yielded chitin dimer (A2) and trimer (A3) [60], which of course could also be

Int. J. Mol. Sci. 2020, 21, 7835 7 of 22

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 22 chitinase yielded chitin dimer (A2) and trimer (A3) [60], which of course could also be generated from thegenerated biotechnologically from the biotechnologically produced pentamer produced using the pentamer recombinant using chitinase the recombinantin vitro. Co-expressingchitinase in vitro. the regio-selectiveCo-expressing chitinthe regio-selective deacetylase NodB chitin from deacetylaseA. caulinodans, NodBwhich from specifically A. caulinodans, deacetylates which thespecifically GlcNAc unitdeacetylates at the non-reducing the GlcNAc end unit of at the the chitin non-reduci oligomer,ng end yielded of the the chitinα-mono-deacetylated oligomer, yielded (‘ αthe’ indicating α-mono- thedeacetylated non-reducing (‘α’ indicating end unit) the chitosan non-reducing pentamer end (A4D1) unit) chit withosan the pentamer sequence (A4D1) DAAAA with [59 the]. sequence Using a GlcN-specificDAAAA [59]. glucosaminidaseUsing a GlcN-specific (GlcNase) glucosaminidase [61], this product (GlcNase) might [61], be this converted product might to yield be theconverted chitin tetramerto yield the (A4) chitin [62]. tetramer (A4) [62].

FigureFigure 4.4. Fully acetylated chitin dimers A2, trimers A3, tetramerstetramers A4,A4, andand pentamerspentamers A5A5 cancan bebe produced biotechnologically in E. coli cell factories on kg scale. An E. coli strain expressing the chitin produced biotechnologically in E. coli cell factories on kg scale. An E. coli strain expressing the chitin oligomer synthase NodC from Rhizobium yields A5. A strain expressing NodC together with NodB oligomer synthase NodC from Rhizobium yields A5. A strain expressing NodC together with NodB yields the α-mono-deacetylated pentamer DAAAA, which can be converted into A4 by GlcNase yields the α-mono-deacetylated pentamer DAAAA, which can be converted into A4 by GlcNase treatment to remove the deacetylated unit from the non-reducing end. A strain co-expressing NodC treatment to remove the deacetylated unit from the non-reducing end. A strain co-expressing NodC and a chitinase produces A3 and A2 which can be separated using chromatography. and a chitinase produces A3 and A2 which can be separated using chromatography. Independent of its route of synthesis, the resulting chitin or glucosamine oligomers will need to Independent of its route of synthesis, the resulting chitin or glucosamine oligomers will need to be purified, to remove oligomers of smaller or larger size than the targeted DP as well as contaminants be purified, to remove oligomers of smaller or larger size than the targeted DP as well as left over from the synthesis. This is particularly crucial if the oligomers are destined to be used later contaminants left over from the synthesis. This is particularly crucial if the oligomers are destined to in bioactivity studies. Purification can best be achieved using chromatographic steps, such as size be used later in bioactivity studies. Purification can best be achieved using chromatographic steps, exclusion chromatography, as detailed at the end of this chapter. Another important issue is the stability such as size exclusion chromatography, as detailed at the end of this chapter. Another important of the oligomers which easily oxidize, visible as a yellowing of the sample. This can be prevented by issue is the stability of the oligomers which easily oxidize, visible as a yellowing of the sample. This storage under anaerobic conditions or in the presence of salt (which, however, may present difficulties can be prevented by storage under anaerobic conditions or in the presence of salt (which, however, in later enzymatic conversion and, particularly, in bioassays) [63]. An added problem for storage arises may present difficulties in later enzymatic conversion and, particularly, in bioassays) [63]. An added for fully or partially de-acetylated oligomers, especially those of low DP, as the free amino groups may problem for storage arises for fully or partially de-acetylated oligomers, especially those of low DP, react with the aldehyde group of the reducing ends of the oligomers, in a Maillard-type reaction which as the free amino groups may react with the aldehyde group of the reducing ends of the oligomers, equally leads to browning of the samples [64]. This can be inhibited by strongly diluting the sample in a Maillard-type reaction which equally leads to browning of the samples [64]. This can be inhibited before freeze drying it. by strongly diluting the sample before freeze drying it. 4. paCOS Nomenclature 4. paCOS Nomenclature In this chapter, we will adopt a widely used, but not yet formalized nomenclature for the descriptionIn thisof chapter, paCOS. we First, will as alreadyadopt a introducedwidely used, above, butwe not will yet abbreviate formalized GlcNAc nomenclature units as ‘A’for andthe GlcNdescription units as of ‘D’. paCOS. Second, First, we as will already use the introduced form ‘AnDm’ above, to indicatewe will theabbreviate number GlcNAc of A and units D units as ‘A’ within and GlcN units as ‘D’. Second, we will use the form ‘AnDm’ to indicate the number of A and D units within the oligomer, without making any statement concerning the sequence of the monomeric units. Thus, ‘AnDm’ includes all paCOS of DP = n + m and FA = n/(n + m). A4D1, e.g., would indicate a

Int. J. Mol. Sci. 2020, 21, 7835 8 of 22

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 8 of 22 the oligomer, without making any statement concerning the sequence of the monomeric units. Thus, paCOS (or a paCOS mixture) of DP 5 and FA 0.8, with no information given on the PA or sequence of ‘AnDm’ includes all paCOS of DP = n + m and FA = n/(n + m). A4D1, e.g., would indicate a paCOS the units. Third, individual positions within the oligomer will be denoted with Greek letters, ‘α‘ (or a paCOS mixture) of DP 5 and FA 0.8, with no information given on the PA or sequence of the units. Third,denoting individual the non-reducing positions end within unit the and oligomer ‘ω’ the willreducing be denoted end unit with of the Greek paCOS. letters, Consequently, ‘α‘ denoting the unitnon-reducing next-to the end non-reducing unit and ‘ω ’end the reducingunit is named end unit ‘β’, of followed the paCOS. by ‘ Consequently,γ’, while the unit the unitnext next-to to the reducingthe non-reducing end unit endis named unit is ‘ψ named’, preceded ‘β’, followed by ‘χ’. Finally, by ‘γ’, to while indicate the unit the fu nextll sequence to the reducing of a paCOS, end unit the unitsis named are listed ‘ψ’, preceded from the bynon-reducing ‘χ’. Finally, to to the indicate reducing the end, full sequencee.g., ‘ADAAA’ of a paCOS, for a β-mono-deacetylated the units are listed pentamericfrom the non-reducing paCOS. As toa consequence, the reducing end,‘AD’ e.g.,indicates ‘ADAAA’ the α for-acetylated a β-mono-deacetylated (or ω-deacetylated) pentameric dimer paCOS.GlcNAc-GlcN, As a consequence, while ‘A1D1’ ‘AD’ summarily indicates denotes the α-acetylated AD and the (or αω-deacetylated-deacetylated) (or dimer ω-acetylated) GlcNAc-GlcN, dimer whileGlcN-GlcNAc, ‘A1D1’ summarily i.e., it can denoteseither indicate AD and one the ofα them-deacetylated (without (or indicatingω-acetylated) which dimer one) or GlcN-GlcNAc, a mixture of bothi.e., it of can them. either indicate one of them (without indicating which one) or a mixture of both of them.

5. Preparation Preparation of paCOS Dimers There are only only two two possible possible dimeric dimeric paCOS, paCOS, name namelyly AD AD and and DA, DA, both both of ofwhich which can can easily easily be producedbe produced from from chitobiose chitobiose (A2) (A2) using using regio-selective, regio-selective, bacterial bacterial CDAs. CDAs. Also, Also, production production of of the substrate chitobiose is comparatively simple, as it is the the main main product product of of chitinase chitinase hydrolysis hydrolysis of of chitin, chitin, particularly if a processive chitinase is used [45,65]. [45,65]. Alternatively, Alternatively, biotechnologically produced chitin pentamer (A5) or preferably, chitinchitin tetramertetramer (A4) maymay be usedused asas aa substrate.substrate. Unless the tetramer is used, thisthis approach approach will will yield yield a mixture a mixture of chitin of dimerchitin anddimer trimer and so trimer that chromatographic so that chromatographic purification purificationis required. is Chitobiose required. canChitobiose be converted can be toconver the dimerted to DAthe dimer by the DA use by of the NodB use fromof NodB any from rhizobial any rhizobialstrain [35 ],strain while [35], the while CDA fromthe CDAVibrio from cholerae Vibrioor choleraeVibrio parahaemolyticus or Vibrio parahaemolyticus, VcCDA or, VcCDA VpCDA, or will VpCDA, yield willthe dimeryield the AD dimer [36,66 ]AD (Figure [36,66]5). (Figure 5).

Figure 5. Biotechnological preparationpreparation of of the the two two dimeric dimeric paCOS paCOS DA DA and and AD. AD. DA canDA becan prepared be prepared from fromchitobiose chitobiose AA by AA the actionby the of ac thetion CDA of NodBthe CDA from NodBRhizobium from. ADRhizobium can be. prepared AD can frombe prepared chitobiose from AA chitobiose AA by the action of VcCDA from Vibrio. The substrate chitobiose AA can be prepared from polymeric chitin or from the chitin pentaose A5 or from the chitin tetraose A4 by the action of a

Int. J. Mol. Sci. 2020, 21, 7835 9 of 22

by the action of VcCDA from Vibrio. The substrate chitobiose AA can be prepared from polymeric chitin or from the chitin pentaose A5 or from the chitin tetraose A4 by the action of a (preferentially processive) chitinase such as SmChiB; the oligomeric substrates being produced by E. coli cell factories expressing the chitin oligomer synthase NodC from Rhizobium to yield A5 or NodC together with NodB to yield the α-mono-deacetylated pentamer DAAAA followed by GlcNase treatment to remove the deacetylated unit from the non-reducing end. In each case, the chitobiose has to be purified using liquid chromatography.

6. Preparation of paCOS Trimers While there are only two possible dimeric paCOS, there are already 23 2 = 6 different trimeric − paCOS which can all be produced using the two bacterial CDAs described above, NodB and VcCDA, which act on the non-reducing α-end unit or the β-unit next to the non-reducing end of their substrates, respectively (Figure6). The fully acetylated substrate A3 can be produced as described above for chitobiose production, namely by chitinase digestion of chitin or biotechnologically produced A5, followed by SEC purification. Similarly, the fully deacetylated glucosamine trimer (D3) can best be produced by chitosanase digestion of polyglucosamine, yielding mostly GlcN dimer (D2) and trimer (D3) which can be separated using SEC [67,68]. There are three different mono-deacetyated trimeric paCOS (A2D1). The trimer DAA can easily be produced from the fully acetylated substrate A3 by the α-action of NodB, while the β-acting VcCDA will yield the trimer ADA when acting on the same substrate [69]. The third, ω-mono-de-acetylated paCOS, AAD, is more difficult to produce as no CDA with regio-selectivity for the GlcNAc unit at the reducing end has been described so far. However, if NodB and VcCDA are used in reverse on the fully deacetylated glucosamine trimer D3 in the presence of excess amounts of acetate, they retain their regio-selectivity, so that their combined use will yield AAD [34]. Similarly, there are three different mono-acetylated paCOS (A1D2) which can be produced in mirror routes to the ones described above. Reverse α-action of NodB on D3 will yield ADD, while reverse β-action of VcCDA will yield DAD [34]. The third, ω-mono-acetylated trimer, DDA, can be produced by the joint forward activity of the two enzymes on the fully acetylated trimer A3 [34]. Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 9 of 22

(preferentially processive) chitinase such as SmChiB; the oligomeric substrates being produced by E. coli cell factories expressing the chitin oligomer synthase NodC from Rhizobium to yield A5 or NodC together with NodB to yield the α-mono-deacetylated pentamer DAAAA followed by GlcNase treatment to remove the deacetylated unit from the non-reducing end. In each case, the chitobiose has to be purified using liquid chromatography.

6. Preparation of paCOS Trimers

While there are only two possible dimeric paCOS, there are already 23 − 2 = 6 different trimeric paCOS which can all be produced using the two bacterial CDAs described above, NodB and VcCDA, which act on the non-reducing α-end unit or the β-unit next to the non-reducing end of their substrates, respectively (Figure 6). The fully acetylated substrate A3 can be produced as described above for chitobiose production, namely by chitinase digestion of chitin or biotechnologically produced A5, followed by SEC purification. Similarly, the fully deacetylated glucosamine trimer (D3) can best be produced by chitosanase digestion of polyglucosamine, yielding mostly GlcN dimer (D2) Int. J. Mol. Sci. 2020, 21, 7835 10 of 22 and trimer (D3) which can be separated using SEC [67,68].

Figure 6. Biotechnological preparation of the six trimeric paCOS DAA, ADA, AAD, DDA, DAD, and ADD. DAA and ADA can be prepared from chitotriose AAA by the action of the CDA NodB from Rhizobium and VcCDA from Vibrio, respectively. DDA can then be produced by subsequent treatment with the other enzyme. Similarly, ADD and DAD can be prepared from DDD by the action of these two enzymes acting in reverse mode in the presence of excess amounts of acetate, and AAD can then be produced by subsequent reverse action of the other enzyme. The substrates for these reactions, AAA and DDD, can be prepared from polymeric chitin or polyglucosamine by the action of a chitinase or chitosanase, respectively. Alternatively, chitotriose can be produced by an E. coli cell factory strain expressing NodC from Rhizobium, yielding chitin pentaose which can then be cleaved, in vivo or in vitro, by a chitinase, yielding chitobiose and chitotriose. In each case, the trimeric product has to be purified using liquid chromatography. 7. Preparation of paCOS Tetramers There are already 24 2 = 14 different possible tetrameric paCOS, and all of them can be produced − using a combination of different CDAs acting in forward or reverse mode [34] (Figure7). Briefly, Int. J. Mol. Sci. 2020, 21, 7835 11 of 22 different CDAs are known which are highly specific in their first deacetylation step so that three of the four mono-deacetylated tetramers are easily produced, only the one with a GlcN unit at its reducing end, AAAD, is more demanding to produce, and the same is true for the production of the mono-acetylated tetramers, using the same enzymes in reverse. All of the six double-acetylated/double-deacetylated tetramers can be produced in not more than two enzymatic steps. In most cases, chromatographic purification is required to obtain pattern-pure tetramers. The tetrameric substrates required, A4 and D4, are more difficult to produce than the smaller substrates required for the production of the dimeric and trimeric paCOS. Both the fully acetylated chitin tetramer A4 and the fully de-acetylated glucosamine tetramer D4 can be produced from tetrameric paCOS generated by the action of chitinase or chitosanase on partially acetylated chitosan polymers, followed by SEC purification. Alternatively, A4 can be produced by GlcNase digestion of the α-mono-deacetylated pentameric paCOS DAAAA, synthesized itself by an E. coli cell factory expressing NodC and NodB from Rhizobium, as described above. There is also an alternative route to the fully de-acetylated tetramer D4 using a mutant of the chitosanase BspCsnMN which has been engineered using site directed mutagenesis, to reduce its ability to hydrolyse the tetramer which consequently is obtained as a product of polyglucosamine digestion, of course also requiring SEC Int.purification J. Mol. Sci. 2020 [70]., 21, x FOR PEER REVIEW 11 of 22

Figure 7. BiotechnologicalBiotechnological preparation preparation of of the the fourteen fourteen possible possible tetrameric tetrameric paCOS paCOS using CDAs in forward or reverse mode on the fully acetylated or or fully de-acetylate de-acetylatedd tetramers AAAA AAAA and and DDDD, DDDD, respectively, asas described previously [[34].34]. Both subs substratestrates can be be prepared prepared biotechnologically biotechnologically in good E. coli yields, AAAA AAAA by by an an E. coli cellcell factory factory expressing expressing NodC NodC and and NodB NodB to toproduce produce DAAAA DAAAA followed followed by GlcNaseby GlcNase treatment treatment to toyield yield AAAA, AAAA, DDDD DDDD by by digest digestionion of of polyglucosamine polyglucosamine using using an an engineered chitosanase unable to cleave tetrameric substrates [70]. Both AAAA and DDDD need to be purified chitosanase unable to cleave tetrameric substrates [70]. Both AAAA and DDDD need to be purified before converting them to paCOS using CDAs. Only five different CDAs are required, acting alone or before converting them to paCOS using CDAs. Only five different CDAs are required, acting alone in sequence, to produce all tetramers. For some of the tetramers, alternative routes exist and typically, or in sequence, to produce all tetramers. For some of the tetramers, alternative routes exist and chromatographic purification is required for the production of pattern-pure paCOS. typically, chromatographic purification is required for the production of pattern-pure paCOS. 8. Preparation of paCOS Pentamers and Hexamers 8. Preparation of paCOS Pentamers and Hexamers There are 25 2 = 30 different pentameric paCOS and 26 2 = 62 different hexameric paCOS. Not all 5 − −6 of themThere can are yet 2 be − produced2 = 30 different using pentameric enzymatic routes.paCOSAlso, and 2 their − 2 = chromatographic 62 different hexameric purification paCOS. is more Not alldemanding of them can and yet still be partly produced impossible. using enzymatic However, routes. a surprisingly Also, their large chromatographic number of paCOS purification pentamers is moreand hexamers demanding can beand produced still partly using impossible. the rather fewHowever, CDAs currentlya surprisingly available large whose number regio-selectivity of paCOS pentamersis known in and suffi hexamerscient detail, can as be shown produced in Figure using8 for th thee rather pentamers. few CDAs While currently it is theoretically available possible whose regio-selectivityto produce all pentamers is known in in this sufficient way using detail, seven as di shownfferent CDAs,in Figure some 8 for will the be dipentamers.fficult to produce While it and is theoreticallytheir yields can possible be expected to produce to be all low, pentamers and most in ofthis them way will using require seven chromatographic different CDAs, some purification. will be difficultAlso, for to two produce of these and enzymes, their yields PcCDA can and be BsPdaCexpected [38 to,39 be], Nlow,-acetylating and most activity of them in thewill presence require chromatographicof excess amounts purification. of acetate has Also, not yetfor beentwo shownof these experimentally, enzymes, PcCDA but is and assumed BsPdaC hypothetically [38,39], N- acetylatinghere. For many activity of the in pentamers, the presence different of productionexcess amounts routes canof acetate be designed has butnot foryet clarity, been onlyshown the experimentally,most feasible one but is is indicated assumed in hypothetically the figure. For here. some For products, many of the alternative pentamers, routes different may giveproduction higher routesyields thancan be the designed route shown but for here, clarity, but weonly opted the most for experimentally feasible one is indicated proven routes in the over figure. theoretically For some products, alternative routes may give higher yields than the route shown here, but we opted for experimentally proven routes over theoretically assumed ones even if the yield of the former is known to be low while that of the latter is expected to be higher. As an example, it is known that AAADD can be produced from A5 by PgtCDA as a by-product in rather low yields (main product is AADDA) [31]. The alternative and possibly higher-yielding production route for this pentamer would start with BsPdaC acting in reverse on D5 and yielding AAAAD, followed by CnCDA4- catalysed de-acetylation to yield AAADD, but the reverse action of BsPDAC has not yet been verified experimentally.

Int. J. Mol. Sci. 2020, 21, 7835 12 of 22 assumed ones even if the yield of the former is known to be low while that of the latter is expected to be higher. As an example, it is known that AAADD can be produced from A5 by PgtCDA as a by-product in rather low yields (main product is AADDA) [31]. The alternative and possibly higher-yielding production route for this pentamer would start with BsPdaC acting in reverse on D5 and yielding AAAAD, followed by CnCDA4-catalysed de-acetylation to yield AAADD, but the reverse action of BsPDACInt. J. Mol. Sci. has 2020 not, 21 yet, x FOR been PEER verified REVIEW experimentally. 12 of 22

Figure 8. Experimentally proven and theoretical production routes for all thirty possible pentameric Figure 8. Experimentally proven and theoretical production routes for all thirty possible pentameric paCOS using known CDAs, alone or in combination, in forward or reverse mode. This overview shows paCOS using known CDAs, alone or in combination, in forward or reverse mode. This overview the most feasible production route for each pentamer, but alternative routes exist for many of them. shows the most feasible production route for each pentamer, but alternative routes exist for many of Since it has not yet been shown that PcCDA and BsPdaC also work in reverse mode, these hypothetical them. Since it has not yet been shown that PcCDA and BsPdaC also work in reverse mode, these steps are marked with an asterisk. hypothetical steps are marked with an asterisk. Overall, the production of the larger oligomers such as the pentamer is more demanding than that of theOverall, tetramers, the and production additional of characteristics the larger oligomers of the di suchfferent as CDAs the pentamer beyond their is more regio-selectivity demanding needthan tothat be of taken the intotetramers, account. and One additional example is characteristics the fact that some of CDAs,the different e.g., VcCDA, CDAs prefer beyond small their substrates regio- overselectivity larger need ones. to In be the taken case into of VcCDA, account. the One mutein exampl VcCDAe is the K275Efact that/H127 some has CDAs, been designede.g., VcCDA, for higherprefer activitysmall substrates on larger over oligomers larger [ 71ones.], and In its the use case is therefore of VcCDA, recommended the mutein inVcCDA the production K275E/H127 of pentameric has been (anddesigned larger) for paCOS. higher Also,activity while on NodBlarger andoligomers VcCDA [71], will and only its de-acetylate use is therefore or N-acetylate recommended a single in unit the evenproduction on larger of pentameric oligomers, CnCDA4 (and larger) and paCOS. PesCDA Also, can deacetylate while NodB further and VcCDA positions, will thus only decreasing de-acetylate the yieldor N-acetylate for their single a single step unit products. even on This larger also oligom decreasesers, theCnCDA4 purity and of the PesCDA product, can since deacetylate the intermediate further productspositions, are thus often decreasing not pattern-pure the yield and for onlytheir if single they di stepffer inproducts. their reducing This also end, decreases they can the be separatedpurity of the product, since the intermediate products are often not pattern-pure and only if they differ in their chromatographically even when having the same FA (see below). For the same reason, also ADDDA andreducing DAAAD end, will they not can be be pattern-pure, separated chroma since theytographically are not the even final when product having of BsPdaC, the same and FA (see they below). cannot (yet)For the be same purified reason, chromatographically also ADDDA and from DAAAD their by-products.will not be pattern-pure, since they are not the final productIn some of BsPdaC, cases, theand sequence they cannot in which (yet) be the puri enzymesfied chromatographically are used also makes from a di fftheirerence by-products. when these largerIn paCOS some cases, with DPthe >sequence4 are targeted. in which As the an enzymes example, are we used mentioned also makes above a difference that three when of the these four mono-deacetylatedlarger paCOS with DP tetramers > 4 are cantargeted. be produced As an exam ratherple, easily, we mentioned since NodB, above VcCDA, that andthree CnCDA4 of the four or PesCDAmono-deacetylated prefer to bind tetramers either can the be reducing produced or therather non-reducing easily, since end NodB, of their VcCDA, substrate and atCnCDA4 a specific or positionPesCDA withinprefer theirto bind substrate either bindingthe reducing groove, or so the that non-reducing specific subsites end thatof their are importantsubstrate forat a substrate specific bindingposition arewithin occupied. their substrate On larger binding oligomers, groove, however, so that other specific effects subsites also play that a role.are important The chitosan for deacetylasesubstrate binding CnCDA4, are foroccupied. example, On starts larger de-acetylating oligomers,the however, fully acetylated other effects pentamer also A5play at thea role. position The nextchitosan to the deacetylase reducing endCnCDA4, of the substrate,for example, yielding starts AAADA.de-acetylating However, the fully as the acetylated enzyme highlypentamer prefers A5 at a the position next to the reducing end of the substrate, yielding AAADA. However, as the enzyme highly prefers a de-acetylated GlcN unit at subsite {−1}, it is impossible to predict whether it would produce ADADA or ADDAA when acting on ADAAA as a substrate [27]. Thus, when targeting the paCOS ADADA, it is preferable to first use CnCDA4 on A5 yielding AAADA followed by VcCDA to yield the targeted ADADA, rather than vice versa. While enzymatic production of all pentameric paCOS is already feasible using the limited number of CDA available which are well enough characterised concerning their precise mode of action, it would highly profit from a few additional CDAs possessing as yet non-described specificities. As an example, the middle position of the pentamer is difficult to de-acetylated or N-

Int. J. Mol. Sci. 2020, 21, 7835 13 of 22 de-acetylated GlcN unit at subsite { 1}, it is impossible to predict whether it would produce ADADA − or ADDAA when acting on ADAAA as a substrate [27]. Thus, when targeting the paCOS ADADA, it is preferable to first use CnCDA4 on A5 yielding AAADA followed by VcCDA to yield the targeted ADADA, rather than vice versa. While enzymatic production of all pentameric paCOS is already feasible using the limited number of CDA available which are well enough characterised concerning their precise mode of action, it would highly profit from a few additional CDAs possessing as yet non-described specificities. As an example, the middle position of the pentamer is difficult to de-acetylated or N-acetylate since none of the CDAs described so far are reported to specifically act on this (γ- or χ-) position. The middle position can only be targeted indirectly using first PcCDA (to yield ADDAA from A5, or DAADD from D5), then VcCDA (converting ADDAA to AADAA in reverse action, or DAADD to DDADD in forward action). If we had a CDA which specifically acted on the middle positions, some of the pentameric paCOS would be easier to produce with better yields. A similar problem occurs for the reducing end, as none of the CDAs described today acts exclusively on that ω-position. Therefore, AAAAD needs to be produced in reverse mode by BsPdaC, a step not yet proven experimentally. As mentioned above, this step would also be a welcome first step in the production of AAADD (by CnCDA4 acting on AAAAD) and ADAAD (by VcCDA acting on AAAAD) in possibly good yields. Of course, as for the shorter paCOS, the fully acetylated and fully de-acetylated substrates A5 and D5 need to be produced first. This can be achieved as described above for the tetramers, i.e., by enzymatic digestion of partially acetylated chitosan polymers followed by chemical N-acetylation or de-acetylation. The chitin pentamer A5 can also be produced biotechnologically in the E. coli cell factory expressing NodC [59], and this A5 might also be fully de-acetylated using alkali to yield D5. As expected, production of defined, pattern-pure paCOS with a DP > 5 is even more demanding. In principle, hexameric paCOS can be produced in the same manner of combining the different CDAs described for the pentameric paCOS production. In this way, more than half of the 62 possible hexameric paCOS can theoretically be produced, although some of them probably only at very low yields. And of course, chromatographic separation will become even more difficult or, rather, impossible. Here, some of the difficulties already described for the production of pentameric paCOS become even more problematic. Again, it is difficult to attack the two middle positions of the hexamer. Let us look at the production of AADAAA. As described by Hembach et al. (2020) [27], ADDAAA can be produced from A6 by the sequential action of the mutein VcCDA K275E/H127 yielding ADAAAA followed by CnCDA4 (or possibly directly by PcCDA acting on A6). Then, ADDAAA needs to be N-acetylated again using the VcCDA mutein to produce the targeted AADAAA. De-acetylation of the other middle position to obtain AAADAA might be possible using PgtCDA as this is the dominating pattern within the mono-deacetylated products of this enzyme. However, PgtCDA very quickly deacetylates a second position, yielding only very low amounts of mono-deacetylated products in general. In addition, as described for the pentameric paCOS, the mixture of mono-deacetylated hexamers cannot be fully separated by HPLC, so that a mixture of A5D1 paCOS will be obtained, with AAADAA being the most prominent, but not the only one. While with the pentamers, the reducing end can be modified using either PgtCDA or BsPdaC in normal or reverse mode, this becomes more challenging with the hexamers, where PgtCDA was not reported to produce AAAADD, but rather AAADDA or AAADDD. It might still be possible to use BsPdaC in reverse mode, though this step has not yet been proven experimentally even on short oligomers, and it can be expected to become more challenging with larger oligomers, since effects such as product inhibition or problems caused by the high salt concentration required for N-acetylation may lead to strongly reduced enzyme activity towards the end of the reaction when the fifth acetate needs to be attached to yield the targeted AAAAAD. Int. J. Mol. Sci. 2020, 21, 7835 14 of 22

9. Mass Spectrometric Analysis of paCOS The structural analysis of paCOS has recently been revolutionized by the use of mass spectrometry (MS) which has a number of advantages over the previously required NMR spectroscopy [32,72,73]. NMR can only solve the sequence of purified paCOS and, concerning full sequencing, is limited to oligomers of low DP 4, as it informs mainly on the α- and ω-end units and their immediately ≤ neighbouring β- and ψ-units. In contrast, mass spectrometry can precisely determine the molecular Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 222 weight of paCOS even in complex mixtures, directly informing about both DP and FA, and MS techniques can then be used to determine PA also, thus giving full structural information. Recently, of oligomers using deuterated acetic anhydride for N-acetylation of paCOS has been described a sophisticated quantitative sequencing method involving isotopic labelling of oligomers using (Figure 9) [32]. deuterated acetic anhydride for N-acetylation of paCOS has been described (Figure9)[32].

Figure 9. Quantitative MS/MS sequencing of a mixture of isotopically labelled chitosan pentamers (A2D3).Figure 9. A Quantitative partially acetylated MS/MS chitosan sequencing polymer of a wasmixture enzymatically of isotopically digested, labelled the free chitosan amino pentamers groups of (A2D3). A partially acetylated chitosan polymer was enzymatically digested, the2 free amino groups GlcN (D) residues within the oligomeric products obtained were acetylated using [ H6]-acetic anhydride, of GlcN (D) 2residues within the oligomeric products obtained were acetylated18 using [2H6]-acetic resulting in [ H3]N-acetylglucosamine (R), and their reducing ends were O-labelled. The labelled 2 18 oligomersanhydride, were resulting separated in [ H using3]N-acetylglucosamine UHPLC hydrophilic (R), interaction and their reducing liquid chromatography ends were O-labelled. (HILIC) andThe analyzedlabelled oligomers using ESI-MS were/MS. separated The sequences using UHPLC (PA) of the hydrophilic chitosan oligomers interaction in theliquid triple-de-acetylated chromatography pentamers(HILIC) and A2D3 analyzed was determinedusing ESI-MS/MS. by using The the sequence relatives abundances,(PA) of the chitosan shown inoligomers the boxes in belowthe triple- the spectrum,de-acetylated of the pentamers fragment A2D3 ions (denoted was determined as B- and by Y-ions using according the relative to nomenclatureabundances, shown created in by the Domon boxes andbelow Costello the spectrum, in 1988 [74 of]), the highlighted fragment in ions brown. (denoted as B- and Y-ions according to nomenclature created by Domon and Costello in 1988 [74]), highlighted in brown. The thus obtained fully re-acetylated chitin oligomers, in which the deuterated acetyl groups markThe the positionsthus obtained of originally fully re-acetylated de-acetylated chitin GlcN oligomers, units in the in paCOS,which the can deuterated then be fragmented acetyl groups and mark the positions of originally de-acetylated GlcN units in the paCOS, can then be fragmented and quantitatively analysed, avoiding problems arising from differences in e.g., fragmentation or ionization of GlcN and GlcNAc units. In this way, the paCOS present in mixtures, e.g., obtained as a result of enzymatic hydrolysis of chitosan polymers or enzymatic de-acetylation or N-acetylation of GlcNAc and GlcN oligomers, respectively, can be identified and quantified, as long as the mixtures are not too complex. The rather low aqueous solubility of chitin oligomers limits this analysis to paCOS with DP ≤ 6. An added advantage of MS over NMR is that the latter requires multiple mg- amounts of sample, while the former can be performed on sub-µg samples.

10. Chromatographic Purification of paCOS

Int. J. Mol. Sci. 2020, 21, 7835 15 of 22 quantitatively analysed, avoiding problems arising from differences in e.g., fragmentation or ionization of GlcN and GlcNAc units. In this way, the paCOS present in mixtures, e.g., obtained as a result of enzymatic hydrolysis of chitosan polymers or enzymatic de-acetylation or N-acetylation of GlcNAc and GlcN oligomers, respectively, can be identified and quantified, as long as the mixtures are not too complex. The rather low aqueous solubility of chitin oligomers limits this analysis to paCOS with DP 6. An added advantage of MS over NMR is that the latter requires multiple mg-amounts of sample, ≤ whileInt. J. Mol. the Sci. former 2020, 21 can, x FOR beperformed PEER REVIEW on sub-µg samples. 15 of 22

10. ChromatographicTypically, both the Purification fully acetylated of paCOS and the fully de-acetylated substrates An and Dn as well as the paCOSTypically, produced both the from fully them acetylated using CDAs and the in fullyforward de-acetylated or reversesubstrates mode need An to and be purified Dn as well using as thechromatography paCOS produced to yield from single them paCOS using [34,69]. CDAs in The forward most straightforward or reverse mode separation need to be is purifiedthat according using chromatographyto DP which can tobe yield achieved single using paCOS size [34 exclusion,69]. The mostchromatography straightforward (SEC) separation [23,69]. isIf thatsufficiently according long to DPcolumns which are can used, be achieved baseline using separation size exclusion of oligomers chromatography up to DP (SEC) ca. 15 [23 can,69 ].be If achieved. sufficiently The long molecular columns areweights used, of baseline the four separation different of dimers oligomers are sufficiently up to DP ca. different 15 can be (DD achieved. = 340 Da, The AD molecular = DA = weights382 Da, ofAA the = four424 Da) different so that dimers they can are be sufficiently partially separated; different (DD the= heaviest340 Da, dimer AD = DA(AA)= is382 sufficiently Da, AA = lighter424 Da than) so that the theylightest can trimer be partially (DDD separated; = 501 Da) theso that heaviest the dimer dimer an (AA)d trimer is sufficiently peaks do lighter not ov thanerlap; the the lightest six different trimer (DDDtrimers= (D3501 Da)= 501 so Da, that A1D2 the dimer = 543 and Da, trimer A2D1 peaks= 585 doDa, not A3 overlap; = 627 Da) the are six not different sufficiently trimers different (D3 = 501 to Da, be A1D2separated= 543 from Da, each A2D1 other.= 585 As Da, a A3consequence,= 627 Da) are chitin not and sufficiently chitosan different oligomers to be (with separated the exception from each of other.dimers) As are a consequence,separated solely chitin according and chitosan to their oligomers DP on currently (with the used exception SEC columns of dimers) (Figure are separated10A). solelyA according disadvantage to their of SEC DP on is that currently typically, used rather SEC columns high salt (Figure concentrations 10A). are needed in the elution bufferA to disadvantage prevent unwanted of SEC iscomplex that typically, formation rather of highthe oligomers salt concentrations and unwanted are needed interactions in the elution of the buoligomersffer to prevent with the unwanted chromatography complex formationmatrix. The of use the oligomersof volatile andsalts unwanted such as ammonium interactions acetate of the oligomersallows to remove with the them chromatography prior to e.g., bioassays, matrix. The bu uset several of volatile sequential salts such freeze-drying as ammonium steps acetate are required allows tofor remove complete them salt prior removal. to e.g., To bioassays,prevent browning but several of the sequential samples, freeze-drying the use of high steps volumes are required of water for is completerequired in salt each removal. step, making To prevent the process browning slow. of the samples, the use of high volumes of water is Fully acetylated chitin oligomers have also been separated by using reverse phase required in each step, making the process slow. chromatography (RPC) [75] or normal phase chromatography (NPC) [76,77]. In contrast to SEC, Fully acetylated chitin oligomers have also been separated by using reverse phase chromatography water can be used as the eluent here, so that no salt removal is required after separation. Fully de- (RPC) [75] or normal phase chromatography (NPC) [76,77]. In contrast to SEC, water can be used as acetylated glucosamine oligomers can also be separated using cation exchange chromatography the eluent here, so that no salt removal is required after separation. Fully de-acetylated glucosamine (CEC), but again with the disadvantage that rather high salt concentrations are required in the eluent oligomers can also be separated using cation exchange chromatography (CEC), but again with the [78]. disadvantage that rather high salt concentrations are required in the eluent [78]. Separating paCOS of equal DP according to their FA is more demanding but can also be achieved, Separating paCOS of equal DP according to their FA is more demanding but can also be achieved, using either CEC, hydrophobic interaction chromatography (HIC), or hydrophilic interaction liquid using either CEC, hydrophobic interaction chromatography (HIC), or hydrophilic interaction liquid chromatography (HILIC). The latter has been shown to even separate some paCOS of equal DP and chromatography (HILIC). The latter has been shown to even separate some paCOS of equal DP and equal FA, but different PA [34,73] (Figure 10B,C). equal FA, but different PA [34,73] (Figure 10B,C).

Figure 10. Cont.

Int. J. Mol. Sci. 2020, 21, 7835 16 of 22 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 22

FigureFigure 10. 10.ChromatographicChromatographic separation separation of different of different paCOS. paCOS. (A) ( ASEC-chromatogram) SEC-chromatogram of the of the separation separation of aof mixture a mixture of chitosans of chitosans ranging ranging from from dimers dimers to polymer to polymer according according to their to their degree degree of polymerisation of polymerisation (DP).(DP). (B) ( BOverlay-HILIC) Overlay-HILIC chromatogram chromatogram of of HILIC-purified HILIC-purified tetrameric tetrameric paCOS paCOS with with different different fractions fractions of of acetylationacetylation ( FA(F).A). (C )( Overlay-HILIC-chromatogramC) Overlay-HILIC-chromatogram of HILIC-purified of HILIC-purified mono-deacetylated mono-deacetylated tetrameric tetramericpaCOS withpaCOS diff witherent different patterns patterns of acetylation of acetylation (PA). (PA). 11. Conclusions and Outlook 11. Conclusions and Outlook Recombinant regio-selective CDAs acting in forward or reverse mode on DP-pure chitin or Recombinant regio-selective CDAs acting in forward or reverse mode on DP-pure chitin or glucosamine oligomers followed by chromatographic purification can yield a plethora of partially glucosamine oligomers followed by chromatographic purification can yield a plethora of partially acetylated chito-oligosaccharides with fully defined architecture in terms of DP, FA, and PA. These can acetylated chito-oligosaccharides with fully defined architecture in terms of DP, FA, and PA. These then be used in a broad range of cellular bio-assays to quantify e.g., their antibacterial, antifungal, can then be used in a broad range of cellular bio-assays to quantify e.g., their antibacterial, antifungal, or orantiviral antiviral activities, activities, their their plant plant growth growth promoting, promoting, disease disease resistance resistance inducing, inducing, and and stress stress tolerance tolerance increasingincreasing activities, activities, their their anti- anti- or or pro-inflammatory,pro-inflammatory, anti-oxidative anti-oxidative or or anti-tumour anti-tumour activities activities and and many manymany many more. more. This This will allowwill allow to fine-tune to fine-tune our current our current understanding understanding of molecular of molecular structure-function structure- functionrelationships relationships of partially of partially acetylated acetylated chitosans, chitosans, but willbut will also also enable enable studies studies on on their their cellular cellular and andphysiological physiological mode mode of action.of action. Clearly, Clearly, such such studies studies will openwill open up a wholeup a whole new dimensionnew dimension of chitosan of chitosanresearch research and development and development and will and pavewill pave the waythe way for third-generationfor third-generation chitosans chitosans with with known known and anddefined defined patterns patterns of of acetylation. acetylation. A A few few studies studies already already hint hint at at the the crucial crucial role role of of PA PA on on properties properties and andbioactivities bioactivities of of chitosans, chitosans, as as recentlyrecently reviewed [6,18], [6,18], and and a avery very recent recent publication publication unequivocally unequivocally α provedproved for for the the first first time time that that the the α-mono-acetylated-mono-acetylated chitosan chitosan tetramer tetramer with with the the GlcNAc GlcNAc unit unit at atits its non-reducingnon-reducing end end had had strong strong disease disease resistance resistance prim priminging activities activities in inrice rice cells, cells, while while the the priming priming ω activitiesactivities sequentially sequentially decreased decreased in inthe the other other three three isomeric isomeric paCOS, paCOS, and and the the ω-mono-acetylated-mono-acetylated one one withwith the the GlcNAc GlcNAc unit unit at at the the reducing endend waswas completely completely inactive inactive [79 [79].]. Importantly, Importantly, this this priming-active priming- activetetramer tetramer possessed possessed no no elicitor elicitor activity, activity, i.e., i.e., it it prepares prepares plantplant cells to to efficiently efficiently react react to to even even low low concentrationsconcentrations of ofa resistance a resistance reaction-inducing reaction-inducing elicit elicitoror that that would would not not trigger trigger a naïve a naïve cell, cell, but but it does it does notnot induce induce such such an energy-consuming an energy-consuming and andpotentially potentially self-destructing self-destructing cellular cellular resistance resistance reaction reaction by itself.by itself.Such an Such elicitor-inactive, an elicitor-inactive, but priming-active but priming-active substance substance is the isideal the lead ideal compound lead compound for the for developmentthe development of a plant of a“vaccination” plant “vaccination” strategy—an strategy—an urgent necessity urgent necessity in our globalised in our globalised world in worldwhich in diseases travel freely and increasingly threaten a safe supply of healthy food and sustainable biomaterials and biologics.

Int. J. Mol. Sci. 2020, 21, 7835 17 of 22 which diseases travel freely and increasingly threaten a safe supply of healthy food and sustainable biomaterials and biologics. Today, not all paCOS can be produced by the enzymatic approach using CDAs. Our improving abilities to heterologously produce these enzymes can be expected to yield more CDAs with differing regio-selectivities, which we can also characterise more precisely given the recent rapid development of analytical tools. This will enable us to make use of nature’s biodiversity in our attempts to provide full sets of fully defined paCOS. At the same time, our increasing knowledge of the structures of CDA enzymes, their subsite specificities or preferences, and their enzymatic modes of action will allow us to design and engineer CDA enzymes with new or more precise regio-selectivities to increase and complement the natural biodiversity [27]. In parallel to these developments, strategies for the chemical synthesis of defined paCOS are also improving, and solid-state synthesis is promising to yield paCOS of every structure at will, even for higher DP, possibly up to DP 10 or even higher [80]. Once these will become available, they will be a more than welcome complementation to the enzymatically produced paCOS. Studies of structure-function relationships have an inherent problem caused by the potentially extremely high bioactivity of signal molecules, necessitating extremely pure test compounds to avoid false conclusions based on bioactivities of minor contaminants. This problem occurs independent of the route of synthesis of the signal compound studied, be it chemical or enzymatic. However, given that possible contaminants can be expected to differ between the two routes, comparison of bioactivities of e.g., paCOS produced by chemical synthesis versus enzymatic modification is the safest way to come to conclusive results, as previously done for other bioactive oligosaccharides [81–84]. The two methods also complement each other in yet another way. While chemical synthesis might eventually turn out to be more flexible in yielding every wanted paCOS, particularly solid-state synthesis cannot easily be scaled to yield larger amounts of products. In contrast, enzymatic synthesis is scalable, both in vitro and in vivo, and cell factories may eventually yield kg amounts of defined paCOS, potentially making the process economically feasible [57]. And finally, we can envisage the development of chemo-enzymatic approaches in which e.g., chemically synthesised paCOS are elongated using glycosyl transferases [85] or joint to yield larger paCOS using glycosyl hydrolases [86] or engineered glyco-synthases [87,88]. The world of third-generation chitosans is only beginning to emerge, but already promises a new era of chitosan research and development of chitosan-based products.

Author Contributions: Conceptualization, M.B., S.S., S.C.-L. and B.M.M.; writing—original draft preparation, M.B., S.C.-L. and B.M.M.; writing—review and editing, M.B., S.S. and S.C.-L.; visualization, M.B.; supervision, S.C.-L. and B.M.M.; project administration, S.S. and S.C.-L.; All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The work of the authors that contributed to this article has been financially supported over the past two decades by national and international grants from e.g., EU, BMBF, BMWi, DFG, DAAD, and DBU. We would like to thank all partners in these collaborative research projects, and particularly the many students, doctoral and post-doctoral researchers who contributed their time and ideas. Above all else, we are deeply indebted to Nour Eddine El Gueddari, our collaborator and friend for twenty years, who suddenly passed away in early 2018. We acknowledge support from the Open Access Publication Fund of the University of Muenster. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

DP Degree of Polymerisation FA Fraction of Acetylation PA Pattern of Acetylation paCOS partially acetylated Chitosan Oligosaccharides CDA Chitin Deacetylase Int. J. Mol. Sci. 2020, 21, 7835 18 of 22

References

1. El Gueddari, N.E.; Moerschbacher, B.M. A bioactivity matrix for chitosans as elicitors of disease resistance reactions in wheat. Adv. Chitin Sci. 2004, VII, 56–59. 2. El Gueddari, N.E.; Kolkenbrock, S.; Schaaf, A.; Chilukoti, N.; Brunel, F.; Gorzelanny, C.; Fehser, S.; Chachra, S.; Bernard, F.; Nampally, M.; et al. Chitin and chitosan modifying enzymes: Versatile novel tools for the analysis of structure-function relationship of partially acetylaed chitosans. Adv. Chitin Sci. 2014, XIV, 40–47. 3. Kauss, H.; Jeblick, W.; Domard, A. The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus. Planta 1989, 178, 385–392. [CrossRef][PubMed] 4. Vander, P.; Vårum, K.M.; Domard, A.; El Gueddari, N.E.; Moerschbacher, B.M. Comparison of the Ability of Partially N-Acetylated Chitosans and Chitooligosaccharides to Elicit Resistance Reactions in Wheat Leaves. Plant Physiol. 1998, 118, 1353–1359. [CrossRef] 5. Moerschbacher, B.M. Bio-activity matrices of chitosans in plant protection. In Emerging Trends in Plant-Microbe Interactions; Gnanamanickam, S.S., Balasubramanian, R., Anand, N., Eds.; University of Madras: Chennai, India, 2005; pp. 186–190. 6. Wattjes, J.; Sreekumar, S.; Richter, C.; Cord-Landwehr, S.; Singh, R.; El Gueddari, N.E.; Moerschbacher, B.M. Patterns matter part 1: Chitosan polymers with non-random patterns of acetylation. React. Funct. Polym. 2020, 151, 104583. [CrossRef] 7. Thadathil, N.; Suresh, P. Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem. 2014, 150, 392–399. [CrossRef] 8. Arnold, N.D.; Brück, W.M.; Garbe, D.; Brück, T.B. Enzymatic Modification of Native Chitin and Conversion to Specialty Chemical Products. Mar. Drugs 2020, 18, 93. [CrossRef] 9. Júnior, E.N.; El Gueddari, N.E.; Moerschbacher, B.M.; Franco, T.T. Growth rate inhibition of phytopathogenic fungi by characterized chitosans. Braz. J. Microbiol. 2012, 43, 800–809. [CrossRef] 10. Lacombe-Harvey, M.È.; Fukamizo, T.; Gagnon, J.; Ghinet, M.G.; Dennhart, N.; Letzel, T.; Brzezinski, R. Accessory active site residues of Streptomyces sp. N174 chitosanase. FEBS J. 2009, 276, 857–869. [CrossRef] 11. Ghinet, M.G.; Roy, S.; Poulin-Laprade, D.; Lacombe-Harvey, M.È.; Morosoli, R.; Brzezinski, R. Chitosanase from Streptomyces coelicolorA3(2): Biochemical properties and role in protection against antibacterial effect of chitosan. Biochem. Cell Biol. 2010, 88, 907–916. [CrossRef] 12. Miya, A.; Albert, P.; Shinya, T.; Desaki, Y.; Ichimura, K.; Shirasu, K.; Narusaka, Y.; Kawakami, N.; Kaku, H.; Shibuya, N. CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc. Natl. Acad. Sci. USA 2007, 104, 19613–19618. [CrossRef][PubMed] 13. Kaku, H.; Nishizawa, Y.; Ishii-Minami, N.; Akimoto-Tomiyama, C.; Dohmae, N.; Takio, K.; Minami, E.; Shibuya, N. Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc. Natl. Acad. Sci. USA 2006, 103, 11086–11091. [CrossRef][PubMed] 14. Gubaeva, E.; Gubaev,A.; Melcher, R.L.J.; Cord-Landwehr, S.; Singh, R.; El Gueddari, N.E.; Moerschbacher, B.M. Slipped Sandwich Model for Chitin and Chitosan Perception in Arabidopsis. Mol. Plant-Microbe Interact. 2018, 31, 1145–1153. [CrossRef][PubMed] 15. Fuchs, K.; Gloria, Y.C.; Wolz, O.; Herster, F.; Sharma, L.; Dillen, C.A.; Täumer, C.; Dickhöfer, S.; Bittner, Z.; Dang, T.; et al. The fungal ligand chitin directly binds TLR 2 and triggers inflammation dependent on oligomer size. EMBO Rep. 2018, 19, e201846065. [CrossRef] 16. Sørbotten, A.; Horn, S.J.; Eijsink, V.G.H.; Vårum, K.M. Degradation of chitosans with chitinase B from Serratia marcescens. FEBS J. 2004, 272, 538–549. [CrossRef] 17. Weikert, T.; Niehues, A.; Cord-Landwehr, S.; Hellmann, M.J.; Moerschbacher, B.M. Reassessment of chitosanase substrate specificities and classification. Nat. Commun. 2017, 8, 1–11. [CrossRef] 18. Cord-Landwehr, S.; Richter, C.; Wattjes, J.; Sreekumar, S.; Singh, R.; Basa, S.; El Gueddari, N.E.; Moerschbacher, B.M. Patterns matter part 2: Chitosan oligomers with defined patterns of acetylation. React. Funct. Polym. 2020, 151, 104577. [CrossRef] 19. Aragunde, H.; Biarnés, X.; Planas, A. Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases. Int. J. Mol. Sci. 2018, 19, 412. [CrossRef] 20. Grifoll-Romero, L.; Pascual, S.; Aragunde, H.; Biarnés, X.; Planas, A. Chitin Deacetylases: Structures, Specificities, and Biotech Applications. Polymers 2018, 10, 352. [CrossRef] Int. J. Mol. Sci. 2020, 21, 7835 19 of 22

21. Zhao, Y.; Park, R.-D.; Muzzarelli, R.A.A. Chitin Deacetylases: Properties and Applications. Mar. Drugs 2010, 8, 24–46. [CrossRef] 22. Tsigos, I.; Martinou, A.; Kafetzopoulos, D.; Bouriotis, V. Chitin deacetylases: New, versatile tools in biotechnology. Trends Biotechnol. 2000, 18, 305–312. [CrossRef] 23. Horn, S.J.; Sorbotten, A.; Synstad, B.; Sikorski, P.; Sorlie, M.; Varum, K.M.; Eijsink, V.G.H. Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens. FEBS J. 2006, 273, 491–503. [CrossRef][PubMed] 24. Fukamizo, T.; Ohkawa, T.; Ikeda, Y.; Goto, S. Specificity of chitosanase from Bacillus pumilus. Biochim. Biophys. Acta (BBA) Protein Struct. Mol. Enzym. 1994, 1205, 183–188. [CrossRef] 25. Hirano, K.; Watanabe, M.; Seki, K.; Ando, A.; Saito, A.; Mitsutomi, M. Classification of Chitosanases by Hydrolytic Specificity toward N1, N4-Diacetylchitohexaose. Biosci. Biotechnol. Biochem. 2012, 76, 1932–1937. [CrossRef] 26. Tokuyasu, K.; Mitsutomi, M.; Yamaguchi, I.; Hayashi, K.; Mori, Y. Recognition of Chitooligosaccharides and TheirN-Acetyl Groups by Putative Subsites of Chitin Deacetylase from a Deuteromycete, Colletotrichum lindemuthianum. Biochemistry 2000, 39, 8837–8843. [CrossRef] 27. Hembach, L.; Bonin, M.; Gorzelanny, C.; Moerschbacher, B.M. Unique subsite specificity and potential natural function of a chitosan deacetylase from the human pathogen Cryptococcus neoformans. Proc. Natl. Acad. Sci. USA 2020, 117, 3551–3559. [CrossRef] 28. Blair, D.E.; Hekmat, O.; Schüttelkopf, A.W.; Shrestha, B.; Tokuyasu, K.; Withers, S.G.; Van Aalten, D.M.F. Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum. Biochemistry 2006, 45, 9416–9426. [CrossRef] 29. Andrés, E.; Albesa-Jové, D.; Biarnés, X.; Moerschbacher, B.M.; Guerin, M.E.; Planas, A. Structural Basis of Chitin Oligosaccharide Deacetylation. Angew. Chem. Int. Ed. 2014, 53, 6882–6887. [CrossRef] 30. Liu, L.; Zhou, Y.; Qu, M.; Qiu, Y.; Guo, X.; Zhang, Y.; Liu, T.; Yang, J.; Yang, Q. Structural and biochemical insights into the catalytic mechanisms of two insect chitin deacetylases of the carbohydrate esterase 4 family. J. Biol. Chem. 2019, 294, 5774–5783. [CrossRef] 31. Naqvi, S.; Cord-Landwehr, S.; Singh, R.; Bernard, F.; Kolkenbrock, S.; El Gueddari, N.E.; Moerschbacher, B.M. A Recombinant Fungal Chitin Deacetylase Produces Fully Defined Chitosan Oligomers with Novel Patterns of Acetylation. Appl. Environ. Microbiol. 2016, 82, 6645–6655. [CrossRef] 32. Cord-Landwehr, S.; Ihmor, P.; Niehues, A.; Luftmann, H.; Moerschbacher, B.M.; Mormann, M. Quantitative Mass-Spectrometric Sequencing of Chitosan Oligomers Revealing Cleavage Sites of Chitosan Hydrolases. Anal. Chem. 2017, 89, 2893–2900. [CrossRef][PubMed] 33. Tokuyasu, K.; Ono, H.; Hayashi, K.; Mori, Y. Reverse hydrolysis reaction of chitin deacetylase and enzymatic synthesis of β-d-GlcNAc-(1 4)-GlcN from chitobiose. Carbohydr. Res. 1999, 322, 26–31. [CrossRef] → 34. Hembach, L.; Cord-Landwehr, S.; Moerschbacher, B.M. Enzymatic production of all fourteen partially acetylated chitosan tetramers using different chitin deacetylases acting in forward or reverse mode. Sci. Rep. 2017, 7, 17692. [CrossRef][PubMed] 35. John, M.; Rohrig, H.; Schmidt, J.; Wieneke, U.; Schell, J. Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase. Proc. Natl. Acad. Sci. USA 1993, 90, 625–629. [CrossRef][PubMed] 36. Li, X.; Wang, L.-X.; Wang, X.; Roseman, S. The Chitin Catabolic Cascade in the Marine Bacterium Vibrio cholerae: Characterization of a Unique Chitin Oligosaccharide Deacetylase. Glycobiology 2007, 17, 1377–1387. [CrossRef] 37. Cord-Landwehr, S.; Melcher, R.L.J.; Kolkenbrock, S.; Moerschbacher, S.; Moerschbacher, B.M. A chitin deacetylase from the endophytic fungus Pestalotiopsis sp. efficiently inactivates the elicitor activity of chitin oligomers in rice cells. Sci. Rep. 2016, 6, 38018. [CrossRef] 38. Aranda-Martinez, A.; Grifoll-Romero, L.; Aragunde, H.; Sancho-Vaello, E.; Biarnés, X.; Lopez-Llorca, L.V.; Planas, A. Expression and specificity of a chitin deacetylase from the nematophagous fungus Pochonia chlamydosporia potentially involved in pathogenicity. Sci. Rep. 2018, 8, 2170. [CrossRef] 39. Grifoll-Romero, L.; Sainz-Polo, M.A.; Albesa-Jové, D.; Guerin, M.E.; Biarnés, X.; Planas, A. Structure-function relationships underlying the dual N-acetylmuramic and N-acetylglucosamine specificities of the bacterial peptidoglycan deacetylase PdaC. J. Biol. Chem. 2019, 294, 19066–19080. [CrossRef] Int. J. Mol. Sci. 2020, 21, 7835 20 of 22

40. No, H.K.; Meyers, S.P. Preperation of chitin and chitosan. In Chitin Handbook; Muzzarelli, R.A.A., Peter, M.G., Eds.; Euopean Chitin Society: Grottammare, Italy, 1997; pp. 475–489. ISBN 88-86889-01-1. 41. Roberts, G.A.F. Chitosan production routes and their role in determining the structure and properties of the product. Adv. Chitin Sci. 1998, II, 22–31. 42. Varum, K. Acid hydrolysis of chitosans. Carbohydr. Polym. 2001, 46, 89–98. [CrossRef] 43. Einbu, A.; Vårum, K.M. Depolymerization and De-N-acetylation of Chitin Oligomers in Hydrochloric Acid. Biomacromolecules 2007, 8, 309–314. [CrossRef][PubMed] 44. Einbu, A.; Grasdalen, H.; Vårum, K.M. Kinetics of hydrolysis of chitin/chitosan oligomers in concentrated hydrochloric acid. Carbohydr. Res. 2007, 342, 1055–1062. [CrossRef] 45. Vaaje-Kolstad, G.; Horn, S.J.; Sørlie, M.; Eijsink, V.G.H. The chitinolytic machinery of Serratia marcescens—A model system for enzymatic degradation of recalcitrant polysaccharides. FEBS J. 2013, 280, 3028–3049. [CrossRef][PubMed] 46. Kidibule, P.E.; Santos-Moriano, P.; Jiménez-Ortega, E.; Ramírez-Escudero, M.; Limón, M.C.; Remacha, M.; Plou, F.J.; Sanz-Aparicio, J.; Fernández-Lobato, M. Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: Enzymatic activity and structural basis of protein specificity. Microb. Cell Factories 2018, 17, 47. [CrossRef][PubMed] 47. Kumirska, J.; Weinhold, M.X.; Steudte, S.; Thöming, J.; Brzozowski, K.; Stepnowski, P. Determination of the pattern of acetylation of chitosan samples: Comparison of evaluation methods and some validation parameters. Int. J. Biol. Macromol. 2009, 45, 56–60. [CrossRef] 48. Vårum, K.M.; Anthonsen, M.W.; Grasdalen, H.; Smidsrød, O. 13C-N.m.r. studies of the acetylation sequences in partially N-deacetylated chitins (chitosans). Carbohydr. Res. 1991, 217, 19–27. [CrossRef] 49. Cord-Landwehr, S.; Niehues, A.; Wattjes, J.; Moerschbacher, B.M. New developments in the analysis of partially acetylated chitosan polymers and oligomers. In Chitin and Chitosan; van den Broek, L.A.M., Boeriu, C.G., Eds.; John Wiley & Sons, Ltd.: Chichester, UK, 2019; pp. 81–95. ISBN 9781119450467. 50. Van Aalten, D.M.F.; Komander, D.; Synstad, B.; Gaseidnes, S.; Peter, M.G.; Eijsink, V.G.H. Structural insights into the catalytic mechanism of a family 18 exo-chitinase. Proc. Natl. Acad. Sci. USA 2001, 98, 8979–8984. [CrossRef] 51. Ohno, T.; Armand, S.; Hata, T.; Nikaidou, N.; Henrissat, B.; Mitsutomi, M.; Watanabe, T. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. J. Bacteriol. 1996, 178, 5065–5070. [CrossRef] 52. Fukamizo, T.; Koga, D.; Goto, S. Comparative Biochemistry of Chitinases—Anomeric Form of the Reaction Products. Biosci. Biotechnol. Biochem. 1995, 59, 311–313. [CrossRef] 53. Sasaki, C.; Vårum, K.M.; Itoh, Y.; Tamoi, M.; Fukamizo, T. Rice chitinases: Sugar recognition specificities of the individual subsites. Glycobiology 2006, 16, 1242–1250. [CrossRef] 54. Regel, E.K.; Weikert, T.; Niehues, A.; Moerschbacher, B.M.; Singh, R. Protein-engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity. Biotechnol. Bioeng. 2018, 115, 863–873. [CrossRef][PubMed] 55. Bußwinkel, F.; Goñi, O.; Cord-Landwehr, S.; O’Connell, S.; Moerschbacher, B.M. Endochitinase 1 (Tv-ECH1) from Trichoderma virens has high subsite specificities for acetylated units when acting on chitosans. Int. J. Biol. Macromol. 2018, 114, 453–461. [CrossRef][PubMed] 56. Aam, B.B.; Heggset, E.B.; Norberg, A.L.; Sørlie, M.; Vårum, K.M.; Eijsink, V.G.H. Production of Chitooligosaccharides and Their Potential Applications in Medicine. Mar. Drugs 2010, 8, 1482–1517. [CrossRef] 57. Naqvi, S.; Moerschbacher, B.M. The cell factory approach toward biotechnological production of high-value chitosan oligomers and their derivatives: An update. Crit. Rev. Biotechnol. 2015, 37, 11–25. [CrossRef] 58. Geremia, R.A.; Mergaert, P.; Geelen, D.; Van Montagu, M.; Holsters, M. The NodC protein of Azorhizobium caulinodans is an N-acetylglucosaminyltransferase. Proc. Natl. Acad. Sci. USA 1994, 91, 2669–2673. [CrossRef] [PubMed] 59. Samain, E.; Drouillard, S.; Heyraud, A.; Driguez, H.; Geremia, R.A. Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli. Carbohydr. Res. 1997, 302, 35–42. [CrossRef] 60. Cottaz, S.; Samain, E. Genetic engineering of Escherichia coli for the production of NI,NII-diacetylchitobiose (chitinbiose) and its utilization as a primer for the synthesis of complex carbohydrates. Metab. Eng. 2005, 7, 311–317. [CrossRef] Int. J. Mol. Sci. 2020, 21, 7835 21 of 22

61. Tanaka, T.; Fukui, T.; Atomi, H.; Imanaka, T. Characterization of an Exo-β-d-Glucosaminidase Involved in a Novel Chitinolytic Pathway from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1. J. Bacteriol. 2003, 185, 5175–5181. [CrossRef] 62. Hamer, S.N.; Moerschbacher, B.M.; Kolkenbrock, S. (Stephan) Enzymatic sequencing of partially acetylated chitosan oligomers. Carbohydr. Res. 2014, 392, 16–20. [CrossRef] 63. Ajandouz, E.H.; Tchiakpe, L.; Ore, F.D.; Benajiba, A.; Puigserver, A. Effects of pH on Caramelization and Maillard Reaction Kinetics in Fructose-Lysine Model Systems. J. Food Sci. 2001, 66, 926–931. [CrossRef] 64. Chung, Y.C.; Tsai, C.-F.; Li, C.F. Preparation and characterization of water-soluble chitosan produced by Maillard reaction. Fish. Sci. 2006, 72, 1096–1103. [CrossRef] 65. Waghmare, S.R.; Ghosh, J.S. Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed. Carbohydr. Res. 2010, 345, 2630–2635. [CrossRef] [PubMed] 66. Kadokura, K.; Sakamoto, Y.; Saito, K.; Ikegami, T.; Hirano, T.; Hakamata, W.; Oku, T.; Nishio, T. Production of a recombinant chitin oligosaccharide deacetylase from Vibrio parahaemolyticus in the culture medium of Escherichia coli cells. Biotechnol. Lett. 2007, 29, 1209–1215. [CrossRef][PubMed] 67. Kurakake, M.; Yo-U, S.; Nakagawa, K.; Sugihara, M.; Komaki, T. Properties of chitosanase from Bacillus cereus S1. Curr. Microbiol. 2000, 40, 6–9. [CrossRef][PubMed] 68. Jung, W.; Park, R.-D. Bioproduction of Chitooligosaccharides: Present and Perspectives. Mar. Drugs 2014, 12, 5328–5356. [CrossRef][PubMed] 69. Hamer, S.N.; Cord-Landwehr, S.; Biarnés, X.; Planas, A.; Waegeman, H.; Moerschbacher, B.; Kolkenbrock, S. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases. Sci. Rep. 2015, 5, 8716. [CrossRef][PubMed] 70. Gercke, D.; Regel, E.; Singh, R.; Moerschbacher, B.M. Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers. J. Biol. Eng. 2019, 13, 23. [CrossRef] 71. Pascual, S.; Planas, A. Screening Assay for Directed Evolution of Chitin Deacetylases: Application to Vibrio cholerae Deacetylase Mutant Libraries for Engineered Specificity. Anal. Chem. 2018, 90, 10654–10658. [CrossRef] 72. Haebel, S.; Bahrke, A.S.; Peter , M.G. Quantitative Sequencing of Complex Mixtures of † Heterochitooligosaccharides by vMALDI-Linear Ion Trap Mass Spectrometry. Anal. Chem. 2007, 79, 5557–5566. [CrossRef] 73. Tang, M.-C.; Nisole, A.; Dupont, C.; Pelletier, J.N.; Waldron, K.C. Chemical profiling of the deacetylase activity of acetyl xylan esterase A (AxeA) variants on chitooligosaccharides using hydrophilic interaction chromatography–mass spectrometry. J. Biotechnol. 2011, 155, 257–265. [CrossRef] 74. Domon, B.; Costello, C.E. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj. J. 1988, 5, 397–409. [CrossRef] 75. Lopatin, S.; Ilyin, M.; Pustobaev, V.; Bezchetnikova, Z.; Varlamov, V.; Davankov, V. Mass-Spectrometric Analysis of N-Acetylchitooligosaccharides Prepared through Enzymatic Hydrolysis of Chitosan. Anal. Biochem. 1995, 227, 285–288. [CrossRef] [PubMed] 76. Li, X.J.; Piao, X.S.; Kim, S.W.; Liu, P.; Wang, L.; Shen, Y.B.; Jung, S.C.; Lee, H.S. Effects of Chito-Oligosaccharide Supplementation on Performance, Nutrient Digestibility, and Serum Composition in Broiler Chickens. Poult. Sci. 2007, 86, 1107–1114. [CrossRef][PubMed] 77. Sashiwa, H.; Fujishima, S.; Yamano, N.; Kawasaki, N.; Nakayama, A.; Muraki, E.; Sukwattanasinitt, M.; Pichyangkura, R.; Aiba, S.-I. Enzymatic production of N-acetyl-d-glucosamine from chitin. Degradation study of N-acetylchitooligosaccharide and the effect of mixing of crude enzymes. Carbohydr. Polym. 2003, 51, 391–395. [CrossRef] 78. Li, K.; Liu, S.; Xing, R.; Yu, H.; Qin, Y.; Li, R.; Li, P. High-resolution separation of homogeneous chitooligomers series from 2-mers to 7-mers by ion-exchange chromatography. J. Sep. Sci. 2013, 36, 1275–1282. [CrossRef] [PubMed] 79. Basa, S.; Nampally, M.; Honorato, T.; Das, S.N.; Podile, A.R.; El Gueddari, N.E.; Moerschbacher, B.M. The Pattern of Acetylation Defines the Priming Activity of Chitosan Tetramers. J. Am. Chem. Soc. 2020, 142, 1975–1986. [CrossRef][PubMed] Int. J. Mol. Sci. 2020, 21, 7835 22 of 22

80. DelBianco, M.; Kononov, A.; Poveda, A.; Yu, Y.; Diercks, T.; Jiménez-Barbero, J.; Seeberger, P.H. Well-Defined Oligo- and Polysaccharides as Ideal Probes for Structural Studies. J. Am. Chem. Soc. 2018, 140, 5421–5426. [CrossRef][PubMed] 81. Darvill, A.G.; Albersheim, P. Phytoalexins and their Elicitors—A Defense Against Microbial Infection in Plants. Annu. Rev. Plant Physiol. 1984, 35, 243–275. [CrossRef] 82. Ossowski, P.; Garegg, P.J.; Lindberg, B. Syntheses of a Branched Hepta and an Octassaccharide with Phytoalexin-Elicitor Activity. Angew. Chem. Int. Ed. 1983, 22, 793–794. [CrossRef] 83. Aly, M.R.; Ibrahim, E.-S.I.; El Ashry, E.S.H.; Schmidt, R.R. Synthesis of chitotetraose and chitohexaose based on dimethylmaleoyl protection. Carbohydr. Res. 2001, 331, 129–142. [CrossRef] 84. Kuyama, H.; Nukada, T.; Ito, Y.; Nakahara, Y.; Ogawa, T.; Nakahara, Y.; Nakahara, Y. Stereocontrolled synthesis of chitosan dodecamer. Carbohydr. Res. 1993, 243, C1–C7. [CrossRef] 85. Slámová, K.; Krejzová, J.; Marhol, P.; Kalachova, L.; Kulik, N.; Pelantová, H.; Cvaˇcka, J.; Kˇren, V. Synthesis of Derivatized Chitooligomers using Transglycosidases Engineered from the Fungal GH20 β-N-Acetylhexosaminidase. Adv. Synth. Catal. 2015, 357, 1941–1950. [CrossRef] 86. Kobayashi, S.; Kiyosada, A.T.; Shoda, S.-I. Synthesis of Artificial Chitin: Irreversible Catalytic Behavior of a Glycosyl Hydrolase through a Transition State Analogue Substrate. J. Am. Chem. Soc. 1996, 118, 13113–13114. [CrossRef] 87. Honda, Y.; Fushinobu, S.; Hidaka, M.; Wakagi, T.; Tsuruta, T.; Taniguchi, H.; Kitaoka, M. Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase. Glycobiology 2008, 18, 325–330. [CrossRef][PubMed] 88. Alsina, C.; Faijes, M.; Planas, A. Glycosynthase-type GH18 mutant chitinases at the assisting catalytic residue for polymerization of chitooligosaccharides. Carbohydr. Res. 2019, 478, 1–9. [CrossRef]

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).